3,4-dihydroxy-2-butanone 4-phosphate synthase

ABSTRACT

Through functional complementation of an Escherichia coli auxotroph, 3,4-dihydroxy-2-butanone 4-phosphate synthase (DS), an indispensable enzyme of the riboflavin biosynthetic pathway of the rice blast fungus Magnaporthe grisea, has been cloned. This invention relates to the isolation of the nucleic acid fragment that encodes the fungal DS protein. In addition, the invention also relates to the construction of chimeric genes encoding all or a portion of the Magnaporthe grisea DS protein, in sense or antisense orientation, wherein the expression of the chimeric gene results in production of altered levels of Magnaporthe grisea DS in a transformed host cell. Finally, the invention also relates the use of the Magnaporthe grisea DS protein as a tool for identifying chemical agents that could be useful as fungicides, antibiotics, or herbicides.

FIELD OF THE INVENTION

This invention is in the field of fungal molecular biology. Morespecifically, this invention pertains to a nucleic acid fragmentencoding a protein involved in the riboflavin biosynthetic pathway offungi.

BACKGROUND OF THE INVENTION

Riboflavin, also referred to as vitamin B₂, is the precursor of flavinmononucleotide (FMN) and flavin adenine dinucleotide (FAD), essentialcofactors for a number of mainstream metabolic enzymes that mediatehydride, oxygen, and electron transfer reactions. Riboflavin-dependentenzymes include succinate dehydrogenase, NADH dehydrogenase,ferredoxin-NADP⁺ oxidoreductase, acyl-CoA dehydrogenase, and thepyruvate dehydrogenase complex. Consequently, fatty acid oxidation, theTCA cycle, mitochondrial electron-transport, photosynthesis, andnumerous other cellular processes are critically dependent on either FMNor FAD as prosthetic groups. Other notable flavoproteins includeglutathione reductase, glycolate oxidase, P450 oxido-reductase, squaleneepoxidase, dihydroorotate dehydrogenase, and a-glycerophosphatedehydrogenase. Genetic disruption of riboflavin biosynthesis inEscherichia coli (Richter et al., J. Bacteriol. 174:4050-4056 (1992))and Saccharomyces cerevisiae (Santos et al., J. Biol. Chem. 270:437444(1995)) results in a lethal phenotype that is only overcome byriboflavin supplementation. This is not surprising, considering theensemble of deleterious pleiotropic effects that would occur withriboflavin deprivation.

Riboflavin is synthesized by plants and numerous microorganisms,including bacteria and fungi (Bacher, A., Chemistry and Biochemistry ofFlavoproteins (Muller, F., ed.) vol. 1, pp. 215-259, Chemical RubberCo., Boca Raton, Fla. (1991)). Since birds, mammals, and other higherorganisms are unable to synthesize the vitamin and, instead, rely on itsdietary ingestion to meet their metabolic needs, the enzymes that areresponsible for riboflavin biosynthesis are favorable targets for futureantibiotics, fungicides, and herbicides as they should have no adverseaffects on such nontarget organisms. Moreover, it is possible that thedistantly-related plant and microbial enzymes have distinctcharacteristics that could be exploited in the development of potentorganism-specific inhibitors. Thus, a detailed understanding of thestructure, mechanism, kinetics, and substrate-binding properties of theriboflavin biosynthetic enzyme(s), from plants for example, would serveas a starting point for the rational design of chemical compounds thatmight be useful as herbicides. Having the authentic fungal or plantprotein(s) in hand would also provide a valuable tool for the in vitroscreening of chemical libraries in search of riboflavin biosynthesisinhibitors.

Fungal and bacterial riboflavin biosynthesis has been intensivelystudied for more than four decades (For recent reviews, see Bacher, A.,Chemistry and Biochemistry of flavoproteins (Muller, F., ed.) vol. 1,pp. 215-259 and 293-316, Chemical Rubber Co., Boca Raton, Fla. (1990)).The synthetic pathway consists of seven distinct enzyme catalyzedreactions, with guanosine 5'-triphosphate (GTP) being the foremostprecursor. ##STR1##

While the second and third steps of riboflavin biosynthesis occur inopposite order in bacteria and fungi, the remaining pathwayintermediates are identical in both microorganisms. Lumazine synthase(LS), the penultimate enzyme of riboflavin biosynthesis, catalyzes thecondensation of 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) with4-ribitylamino-5-amino-2,6-dihydroxypyrimidine (RAADP) to yield 1 moleach of orthophosphate and 6,7-dimethyl-8-(1'-D-ribityl)-lumazine(DMRL), the immediate precursor of riboflavin. The terminal step ofriboflavin biosynthesis is mediated by riboflavin synthase (RS). Thisenzyme catalyzes the dismutation of two molecules of MRL to yield 1 molof riboflavin and RAADP.

Ribulose 5-phosphate serves as substrate for the formation of DHBPcatalyzed by the enzyme 3,4-dihydroxy-2-butanone 4-phosphate synthase(DS). The complex enzyme reaction involving DS entails the eliminationof C-4 from Ribulose 5-phosphate as formate via an intramolecularrearrangement as well as the conversion of the position 1 hydroxymethylgroup to a methyl group. The catalytic process probably involves asequence of tautomerization reactions. It is remarkable that such acomplex reaction can be performed by a single and relatively smallprotein!

DS-encoding genes have been cloned from numerous organisms, includingEscherichia coli (GenBank accession number X66720; Richter et al., J.Bacteriol. 174:40504056 (1992)), Vibrio harvey (GenBank accession numberM27139; Swartztan et al., J. Biol. Chem. 265:3513-3517 (1989)),Photobacterium phosphoreum (GenBank accession number L11391; Lee et al.,J. Bacteriol. 176:2100-2104 (1994)), Bacillus substilis (GenBankaccession number X51510; Kil et al., Mol. Gen. Genet. 233:483-486(1992)), Bacillus amyloliquefaciens (GenBank accession number X95955;Gusarov et al., Mol. Biol. 31:370-376 (1997)), Actinobacilluspleuropneumoniae (GenBank accession number U27202; Fuller et al., J.Bacteriol. 177:7265-7270 (1995)), Saccharomyces cerevisiae (GenBankaccession number Z21619; Revuelta, J. L., direct submission; WO 9411515)and Ashbya gossypii (DGene accession number 95N-T03516; DE 4420785).While the various DS homologs all share certain structural features incommon, their overall homology at the primary amino acid level is ratherpoor. For example, as determined with the Genetics Computer Group Gapprogram (Wisconsin Package Version 9.0, Genetics Computer Group (GCG),Madison, Wis. using their standard default values for "gap creationpenalty" of 12 and "gap extension penalty" of 4), the Escherichia coliis only 61%, 25%, 16%, 27%, 33%, 45% and 43% identical to the homologousproteins of Vibrio harveyi, Photobacterium phosphoreum, Bacillussubstilis, Bacillus amyloliquefaciens, Actinobacillus pleuropneumoniae,Saccharomyces cerevisiae and Ashbya gossypii, respectively. In addition,pairwise comparisons of these eight proteins reveal that the two mostsimilar homologs share only 61% identity. The only known isolated fungalDS genes are that of Ashbya gossypii and Saccharomyces cerevisiae.

From the foregoing discussion, it is apparent that too little is knownabout fungal DS genes/proteins and their relationship to known microbialhomologs to allow isolation of DS-encoding genes from any fungal orplant species using most classical approaches. The latter includehybridization probing of cDNA libraries with homologous or heterologousgenes, PCR-amplification of the gene of interest using oligionucleotideprimers corresponding to conserved amino acid sequence motifs, and/orimmunological detection of expressed cDNA inserts in microbial hosts.Unfortunately, these techniques would not be expected to be very usefulfor the isolation of fungal or plant DS genes, since they all heavilyrely on the presence of significant structural similarity (i.e., DNA oramino acid sequence) with known proteins and genes that have the samefunction. Given the observation that DS proteins are so poorlyconserved, even amongst microorganisms, it is highly unlikely that theknown microbial homologs would share significant structural similaritieswith their counterparts in higher plants.

An alternative approach that has been used to clone biosynthetic genesin other metabolic pathways from higher eucaryotes is throughcomplementation of microbial mutants that are deficient in the enzymeactivity of interest. Since this strategy relies only on the functionalsimilarity between the disrupted host protein and the target gene ofinterest, it is ideally suited for cloning structurally dissimilarproteins that catalyze the same reaction. For functionalcomplementation, a cDNA library is constructed in a vector that candirect the expression of the cDNA in the microbial host. The plasmidlibrary is then introduced into the mutant microbe, and colonies areselected that are no longer phenotypically mutant. Indeed, thearabidopsis GTP cyclohydrolase II (Kobayashi et al, Gene 160:303-304(1995)), LS (Garcia-Ramirez et al., J. Biol. Chem. 270:23801-23807(1995)) and RS (Santos et al., J. Biol. Chem. 270:437444 (1995)) ofyeast, were all cloned through functional complementation of microbialriboflavin auxotrophs. This strategy has also worked for isolating genesfrom higher eucaryotes that are involved in other metabolic pathways,including lysine biosynthesis (Frisch et al., Mol. Gen. Genet.228:287-293 (1991)), purine biosynthesis (Aimi et al., J. Biol. Chem.265:9011-9014 (1990)), and tryptophan biosynthesis (Niyogi et al., PlantCell 5:1011-1027 (1993)), and has also been successfully employed in theisolation of various plant genes including glutamine synthetase (Snustadet al., Genetics 120:1111-1124 (1988)), pyrroline-5-carboxylatereductase (Delauney et al., Mol. Genet. 221:299-305 (1990)),dihydrodipicolinate synthase (Frisch et al., Mol. Gen. Genet.228:287-293 (1991)), 3-isopropylmalate dehydrogenase (Ellerstrom et al.,Plant Mol. Biol. 18:557-566 (1992)), and dihydroorotate dehydrogenase(Minet et al., Plant J. 2:417422 (1992)).

Despite the obvious attractive features of cloning by functionalcomplementation, there are several reasons why this approach might notwork when applied to a fungal DS gene. First, the fungal cDNA sequencemight not be expressed at adequate levels in the mutant microbe for avariety of reasons, including differences in preferred codon usage.Second, the cloned DS gene might not produce a functional polypeptide,if for instance, enzyme activity requires a post-translationalmodification, such as acetylation, glycosylation, or phosphorylationthat is not carried out by the microbial host. Third, the heterologousfungal protein might be lethal to the host, thus rendering itsexpression impossible. Fourth, the fungal protein might fail to achieveits native conformation in the foreign microbial environment, due tofolding problems, inclusion body formation, or various other reasons. Ifany of these events were to occur, cloning the DS gene by functionalcomplementation would not be possible.

SUMMARY OF THE INVENTION

The instant invention relates to an isolated nucleic acid fragmentencoding an indespensible fungal enzyme involved in riboflavinbiosynthesis. Specifically, this invention concerns an isolated nucleicacid fragment encoding a fungal DS, wherein the fungus is Magnaporthegrisea. In addition, this invention relates to nucleic acid fragmentsthat are complementary to a nucleic acid fragment encoding a fungal DSenzyme.

In another embodiment, the instant invention relates to chimeric genesencoding a fungal DS enzyme or to chimeric genes that comprise nucleicacid fragments that are complementary to the nucleic acid fragmentencoding the enzyme, operably linked to suitable regulatory sequences,wherein expression of the chimeric genes results in production of levelsof the encoded enzymes in transformed host cells that are altered (i.e.,increased or decreased) from the levels produced in the untransformedhost cells.

In a further embodiment, the instant invention concerns a transformedhost cell comprising in its genome a chimeric gene encoding a fungal DSenzyme, operably linked to suitable regulatory sequences, whereinexpression of the chimeric gene results in production of altered levelsof a fungal DS enzyme in the transformed host cell. The transformed hostcells can be of eucaryotic or procaryotic origin, and include cellsderived from higher plants or microorganisms. The invention alsoincludes transformed plants that arise from transformed host cells ofhigher plants, and from seeds derived from such transformed plants.

An additional embodiment of the instant invention, concerns a method ofaltering the level of expression of a fungal DS enzyme in a transformedhost cell comprising: a) transforming a host cell with the chimeric geneencoding a fungal DS enzyme, operably linked to suitable regulatorysequences; and b) growing the transformed host cell under conditionsthat are suitable for expression of the chimeric gene wherein expressionof the chimeric gene results in production of altered levels of DS inthe transformed cell.

An additional embodiment of the instant invention concerns a method forobtaining a nucleic acid fragment encoding all or substantially all ofan amino acid sequence encoding a fungal DS.

Additionally, a method is provided for evaluating at least one compoundfor its ability to inhibit the activity of a fungal DS enzyme and thusserve as a crop protection chemical comprising the steps of: (a)transforming a host cell with a chimeric gene comprising an isolatednucleic acid fragment encoding a fungal DS enzyme, the chimeric geneoperably linked to suitable regulatory sequences; (b) growing thetransformed host cell of step (a) under conditions suitable forexpression of the chimeric gene wherein the expression of the chimericgene results in the production of the fungal DS enzyme; (c) contactingthe transformed host cell with a chemical compound; and (d) comparingthe metabolic activity of the transformed host cell that has beencontacted with the chemical compound with the metabolic activity of anuntreated host cell, a decrease in metabolic activity in the contactedtransformed host cell as compared to the untreated transformed host cellin step (d) indicating that the chemical compound is potentially usefulas a crop protection chemical.

In an alternate embodiment, a method is provided for evaluating at leastone compound for its ability to inhibit the activity of a fungal DSenzyme and thus serve as a crop protection chemical comprising the stepsof: (a) transforming a host cell with a chimeric gene comprising anucleic acid fragment encoding a fungal DS enzyme, the chimeric geneoperably linked to suitable regulatory sequences; (b) growing thetransformed host cell of step (a) under conditions suitable forexpression of the chimeric gene wherein the expression of the chimericgene results in the production of the fungal DS enzyme; (c) optionallypurifying the enzyme expressed by the transformed host cell; (d)contacting the enzyme with a chemical compound; and (e) comparing theactivity of the enzyme that has been contacted with the test compound tothe activity of the untreated enzyme, a decrease in activity of thecontacted enzyme as compared to the activity level of the untreatedenzyme in step (e) indicating that the chemical compound is potentiallyuseful as a crop protection chemical.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

FIG. 1 shows a primary amino acid sequence alignment of known microbialDS homologs and the cloned Magnaporthe grisea DS protein that wasgenerated with the GCG Pileup program (Genetics Computer Group, Madison,Wis.).

The following sequence descriptions and sequence listings attachedhereto comply with the rules governing nucleotide and/or amino acidsequence disclosures in patent applications as set forth in 37 C.F.R.§1.821-1.825. The Sequence Descriptions contain the one letter code fornucleotide sequence characters and the three letter codes for aminoacids as defined in conformity with the IUPAC-IYUB standards describedin Nucleic Acids Research 13:3021-3030 (1985) and in the BiochemicalJournal 219(2):345-373 (1984) which are herein incorporated byreference. The present invention utilized Wisconsin Package Version 9.0software from Genetics Computer Group (GCG), Madison, Wis. using theirstandard default values for "gap creation penalty" of 12 and "gapextension penalty" of 4. Gap uses the algorithm of Needleman and Wunschto find the alignment of two complete sequences that maximizes thenumber of matches and minimizes the number of gaps.

SEQ ID NO:1 is the nucleotide sequence of a cloned cDNA encodingMagnaporthe grisea DS.

SEQ ID NO:2 is the deduced amino acid sequence of the cloned cDNAencoding Magnaporthe grisea DS.

SEQ ID NO:3 is the 5' primer useful in the amplification of E. coli DShaving GenBank accession number X66720.

SEQ ID NO:4 is the 3' primer useful in the amplification of E. coli DShaving GenBank accession number X66720.

SEQ ID NO:5 is the 5' primer useful for the introduction of a DNAfragment that confers kanamycin resistance into the E. coli DS genehaving GenBank accession number X66720, respectively, at a NotI cleavagesite.

SEQ ID NO:6 is the 3' primer useful for the introduction of a DNAfragment that confers kanamycin resistance into the E. coli DS genehaving GenBank accession number X66720, respectively, at a NotI cleavagesite.

SEQ ID NO:7 is one of the PCR primers useful for the introduction of aNotI cleavage site in the middle of E. coli DS having GenBank accessionnumber X66720. (hybridizes to nt 968-987).

SEQ ID NO:8 is one of the PCR primers useful for the introduction of aNotI cleavage site in the middle of E. coli DS having GenBank accessionnumber X66720. (hybridizes to nt 940-957).

SEQ ID NO:9 is the 5' primer useful for introducing Magnaporthe griseaDS into the E. coli expression vector, pET-24a (+) (Novagen).

SEQ ID NO:10 is the 3' primer useful for introducing Magnaporthe griseaDS into the E. coli expression vector, pET-24a (+) (Novagen).

DETAILED DESCRIPTION OF THE INVENTION

3,4-Dihydroxy-2-butanone 4-phosphate synthase (DS) of the Magnaporthegrisea riboflavin biosynthetic pathway has been cloned by functionalcomplementation of an Escherichia coli auxotroph.

A nucleic acid fragment that encodes the DS protein has been isolatedfrom Magnaporthe grisea. The invention also includes an assay using theprotein that is encoded for by the nucleic acid fragment to screen forcrop protection chemicals (e.g., fungicides) related to the fungalriboflavin biosynthetic pathway.

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions are provided.

"3,4-Dihydroxy-2-butanone 4-phosphate synthase" is abbreviated as DS,and refers to the enzyme that catalyzes the conversion of ribulose5-phosphate to 3,4-dihydroxy-2-butanone 4-phosphate and formic acid.

"Polymerase chain reaction" is abbreviated PCR.

"Expressed sequence tag" is abbreviated EST.

"3,4-Dihydroxy-2-butanone 4-phosphate" is abbreviated DHBP.

"Isopropyl-1-thio-β-D-galactopyranoside" is abbreviated IPTG.

"Sodium dodecylsulfate-polyacrylamide gel electrophoresis" isabbreviated SDS-PAGE.

"Open reading frame" is abbreviated ORF.

An "isolated nucleic acid fragment" is a polymer of RNA or DNA that issingle- or double-stranded, optionally containing synthetic, non-naturalor altered nucleotide bases. An isolated nucleic acid fragment in theform of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA or synthetic DNA.

"Auxotrophy" refers to the nutritional requirements necessary forgrowth, sporulation and crystal production of the microorganism. For thepurpose of this invention, the term "auxotroph" is defined herein tomean an organism which requires the addition of riboflavin for growth.

The terms "host cell" and "host organism" refer to a cell capable ofreceiving foreign or heterologous genes and expressing those genes toproduce an active gene product. Suitable host cells includemicroorganisms such as bacteria and fungi, as well as plant cells.

The term "metabolic activity" refers to the normal cellular activityneeded to support growth. As used herein agents, such as crop protectionchemicals, that will inhibit metabolic activity will generally inhibitcell growth.

The term, "substantially similar" refers to nucleic acid fragmentswherein changes in one or more nucleotide bases result in substitutionof one or more amino acids, but do not affect the functional propertiesof the protein encoded by the DNA sequence. "Substantially similar" alsorefers to nucleic acid fragments wherein changes in one or morenucleotide bases do not affect the ability of the nucleic acid fragmentto mediate alteration of gene expression by antisense or co-suppressiontechnology. "Substantially similar" also refers to modifications of thenucleic acid fragments of the instant invention such as deletion orinsertion of one or more nucleotide bases that do not substantiallyaffect the functional properties of the resulting transcript vis-a-visthe ability to mediate alteration of gene expression by antisense orco-suppression technology or alteration of the functional properties ofthe resulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary sequences.

A "substantial portion" refers to an amino acid or nucleotide sequencewhich comprises enough of the amino acid sequence of a polypeptide orthe nucleotide sequence of a gene to afford putative identification ofthat polypeptide or gene, either by manual evaluation of the sequence byone skilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Basic Local AlignmentSearch Tool; Altschul et al., J. Mol. Biol. 215:403-410 (1990); see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence often or morecontiguous amino acids or thirty or more nucleotides is necessary inorder to putatively identify a polypeptide or nucleic acid sequence ashomologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides (generally 12 bases orlonger) may be used as amplification primers in PCR in order to obtain aparticular nucleic acid fragment comprising the primers. Accordingly, a"substantial portion" of a nucleotide sequence comprises enough of thesequence to afford specific identification and/or isolation of a nucleicacid fragment comprising the sequence. The instant specification teachespartial or complete amino acid and nucleotide sequences encoding one ormore particular fungal proteins. The skilled artisan, having the benefitof the sequences as reported herein, may now use all or a substantialportion of the disclosed sequences for the purpose known to thoseskilled in the art. Accordingly, the instant invention comprises thecomplete sequences as reported in the accompanying Sequence Listing, aswell as substantial portions of those sequences as defined above.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby nucleic acid fragments that do not share 100% identity with the geneto be suppressed. Moreover, alterations in a gene which result in theproduction of a chemically equivalent amino acid at a given site, but donot effect the functional properties of the encoded protein, are wellknown in the art. Thus, a codon for the amino acid alanine, ahydrophobic amino acid, may be substituted by a codon encoding anotherless hydrophobic residue, such as glycine, or a more hydrophobicresidue, such as valine, leucine, or isoleucine. Similarly, changeswhich result in substitution of one negatively charged residue foranother, such as aspartic acid for glutamic acid, or one positivelycharged residue for another, such as lysine for arginine, can also beexpected to produce a functionally equivalent product.

Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the protein molecule would also not be expectedto alter the activity of the protein. Each of the proposed modificationsis well within the routine skill in the art, as is determination ofretention of biological activity of the encoded products. Moreover, theskilled artisan recognizes that substantially similar sequencesencompassed by this invention are also defined by their ability tohybridize, under stringent conditions (0.1× SSC, 0.1% SDS, 65° C.), withthe sequences exemplified herein. Preferred substantially similarnucleic acid fragments of the instant invention are those nucleic acidfragments whose DNA sequences are 80% identical to the DNA sequence ofthe nucleic acid fragments reported herein. More preferred nucleic acidfragments are 90% identical to the DNA sequence of the nucleic acidfragments reported herein. Most preferred are nucleic acid fragmentsthat are 95% identical to the DNA sequence of the nucleic acid fragmentsreported herein.

"Codon degeneracy" refers to redundancy in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment that encodes all or a substantialportion of the amino acid sequence encoding the DS biosynthetic enzymeas set forth in SEQ ID NO:2. The skilled artisan is well aware of the"codon-bias" exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. Therefore, when synthesizing agene for improved expression in a host cell, it is desirable to designthe gene such that its frequency of codon usage approaches the frequencyof preferred codon usage of the host cell.

The term "percent identity", as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,"identity" also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. "Identity"and "similarity" can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, New York (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, New York (1993); Computer Analysis of Sequence Data PartI (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey(1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.)Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. andDevereux, J., eds.) Stockton Press, New York (1991). Preferred methodsto determine identity are designed to give the largest match between thesequences tested. Methods to determine identity and similarity arecodified in publicly available computer programs. Preferred computerprogram methods to determine identity and similarity between twosequences include, but are not limited to, the GCG Pileup program foundin the GCG program package, as used in the instant invention, using theNeedleman and Wunsch algorithm with their standard default values of gapcreation penalty=and gap extension penalty=4 (Devereux et al., NucleicAcids Res. 12:387-395 (1984)), BLASTP, BLASTN, and FASTA (Pearson etal., Proc. Natl. Acad. Sci. U.S.A. 85:2444-2448 (1988). The BLAST Xprogram is publicly available from NCBI and other sources (BLAST Manual,Altschul et al., Natl. Cent. Biotechnol. Inf., Natl. Library Med. (NCBINLM) NIH, Bethesda, Md. 20894; Altschul et al., J. Mol. Biol.215:403-410 (1990)). Another preferred method to determine percentidentity, is by the method of DNASTAR protein alignment protocol usingthe Jotun-Hein algorithm (Hein et al., Methods Enzymol. 183:626-645(1990)). Default parameters for the Jotun-Hein method for alignmentsare: for multiple alignments, gap penalty=11, gap length penalty=3; forpairwise alignments ktuple=6. As an illustration, by a polynucleotidehaving a nucleotide sequence having at least, for example, 95%"identity" to a reference nucleotide sequence of SEQ ID NO:1 it isintended that the nucleotide sequence of the polynucleotide is identicalto the reference sequence except that the polynucleotide sequence mayinclude up to five point mutations per each 100 nucleotides of thereference nucleotide sequence of SEQ ID NO:1. In other words, to obtaina polynucleotide having a nucleotide sequence at least 95% identical toa reference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence.These mutations of the reference sequence may occur at the 5' or 3'terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence. Analogously, by a polypeptidehaving an amino acid sequence having at least, for example, 95% identityto a reference amino acid sequence of SEQ ID NO:2 is intended that theamino acid sequence of the polypeptide is identical to the referencesequence except that the polypeptide sequence may include up to fiveamino acid alterations per each 100 amino acids of the reference aminoacid of SEQ ID NO:2. In other words, to obtain a polypeptide having anamino acid sequence at least 95% identical to a reference amino acidsequence, up to 5% of the amino acid residues in the reference sequencemay be deleted or substituted with another amino acid, or a number ofamino acids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

The term "complementary" is used to describe the relationship betweennucleotide bases that are capable of hybridizing to one another. Hencewith respect to DNA, adenosine is complementary to thymine and cytosineis complementary to guanine.

"Synthetic genes" can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments which are then enzymatically assembled to construct the entiregene. "Chemically synthesized", as related to a sequence of DNA, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of nucleotidesequence to reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

"Gene" refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5' non-codingsequences) and following (3' non-coding sequences) the coding sequence."Native gene" refers to a gene as found in nature with its ownregulatory sequences. "Chimeric gene" refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. "Endogenous gene" refers to a native gene in its naturallocation in the genome of an organism. A "foreign" gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A "transgene" isa gene that has been introduced into the genome by a transformationprocedure.

"Coding sequence" refers to a DNA sequence that codes for a specificamino acid sequence. "Regulatory sequences" refer to nucleotidesequences located upstream (5' non-coding sequences), within, ordownstream (3' non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

"Promoter" refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3' to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an "enhancer" is aDNA sequence which can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. Promoters whichcause a gene to be expressed in most cell types at most times arecommonly referred to as "constitutive promoters". New promoters ofvarious types useful in plant cells are constantly being discovered;numerous examples may be found in the compilation by Okamuro andGoldberg, (Biochemistry of Plants 15:1-82 (1989)). It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of differentlengths may have identical promoter activity.

The "translation leader sequence" refers to a DNA sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed MRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, MRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner et al., Mol. Biotech. 3:225(1995)).

The "3' non-coding sequences" refer to DNA sequences located downstreamof a coding sequence and include polyadenylation recognition sequencesand other sequences encoding regulatory signals capable of affectingMRNA processing or gene expression. The polyadenylation signal isusually characterized by affecting the addition of polyadenylic acidtracts to the 3' end of the mRNA precursor. The use of different 3'non-coding sequences is exemplified by Ingelbrecht et al., Plant Cell1:671-680 (1989).

"RNA transcript" refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. "Messenger RNA" (MRNA) refers tothe RNA that is without introns and that can be translated into proteinby the cell. "cDNA" refers to a double-stranded DNA that iscomplementary to and derived from mRNA. "Sense" RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. "Antisense RNA" refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or MRNA andthat blocks the expression of a target gene (U.S. Pat. No. 5,107,065).The complementarity of an antisense RNA may be with any part of thespecific gene transcript, i.e., at the 5' non-coding sequence, 3'non-coding sequence, introns, or the coding sequence. "Functional RNA"refers to antisense RNA, ribozyme RNA, or other RNA that is nottranslated yet has an effect on cellular processes.

The term "operably linked" refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term "expression", as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of MRNA into a polypeptide. "Antisense inhibition" refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. "Overexpression" refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. "Co-suppression"refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020).

"Altered levels" refers to the production of gene product(s) inorganisms in amounts or proportions that differ from that of normal ornon-transformed organisms.

"Transformation" refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as "transgenic" organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al., Meth. Enzymol. 143:277 (1987)) andparticle-accelerated or "gene gun" transformation technology (Klein etal., Nature, London 327:70-73 (1987); US 4,945,050).

A Magnaporthe grisea DS has been isolated and identified by comparisonof random cDNA sequences to the GenBank database using the BLASTalgorithms well known to those skilled in the art. The nucleotidesequence of Magnaporthe grisea DS cDNA is provided in SEQ ID NO:1, andthe deduced amino acid sequence is provided in SEQ ID NO:2. A DS genefrom other fungi can now be identified by comparison of random cDNAsequences to the Magnaporthe grisea DS sequence provided herein.

The nucleic acid fragment of the instant invention may be used toisolate cDNAs and genes encoding a homologous DS from the same or otherfungal species. Isolation of homologous genes using sequence-dependentprotocols is well known in the art. Examples of sequence-dependentprotocols include, but are not limited to, methods of nucleic acidhybridization, and methods of DNA and RNA amplification as exemplifiedby various uses of nucleic acid amplification technologies (e.g.,polymerase chain reaction (PCR) or ligase chain reaction).

For example, DS genes, either as cDNAs or genomic DNAs, could beisolated directly by using all or a portion of the instant nucleic acidfragments as DNA hybridization probes to screen libraries from anydesired fungus employing methodology well known to those skilled in theart. Specific oligonucleotide probes based upon the instant DS sequencecan be designed and synthesized by methods known in the art (Maniatisinfra). Moreover, the entire sequences can be used directly tosynthesize DNA probes by methods known to the skilled artisan such asrandom primers, DNA labeling, nick translation, or end-labelingtechniques, or RNA probes using available in vitro transcriptionsystems. In addition, specific primers can be designed and used toamplify a part of or full-length of the instant sequences. The resultingamplification products can be labeled directly during amplificationreactions or labeled after amplification reactions, and used as probesto isolate full length cDNA or genomic fragments under conditions ofappropriate stringency.

In addition, two short segments of the instant nucleic acid fragment maybe used in PCR protocols to amplify longer nucleic acid fragmentsencoding homologous DS genes from DNA or RNA. The polymerase chainreaction may also be performed on a library of cloned nucleic acidfragments wherein the sequence of one primer is derived from the instantnucleic acid fragment, and the sequence of the other primer takesadvantage of the presence of the polyadenylic acid tracts to the 3' endof the MRNA precursor encoding fungal DS. Alternatively, the secondprimer sequence may be based upon sequences derived from the cloningvector. For example, the skilled artisan can follow the RACE protocol(Frohman et al., Proc. Natl. Acad. Sci., USA 85:8998 (1988)) to generatecDNAs by using PCR to amplify copies of the region between a singlepoint in the transcript and the 3' or 5' end. Primers oriented in the 3'and 5' directions can be designed from the instant sequences. Usingcommercially available 3' RACE or 5' RACE systems (BRL), specific 3' or5' cDNA fragments can be isolated (Ohara et al., Proc. Natl. Acad. Sci.,USA 86:5673 (1989); Loh et al., Science 243:217 (1989)). Productsgenerated by the 3' and 5' RACE procedures can be combined to generatefull-length cDNAs (Frohman et al., Techniques 1:165 (1989)).

Finally, availability of the instant nucleotide and deduced amino acidsequence facilitates immunological screening cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequence may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequence. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner et al., Adv.Immunol. 36:1 (1984); Maniatis infra).

The nucleic acid fragments of the instant invention may also be used tocreate transgenic plants in which the instant DS protein is present athigher or lower levels than normal. Such manipulations would conceivablyalter the intracellular levels of riboflavin, hence the essentialcofactors FAD and FMN, producing novel phenotypes of potentialcommercial value. Alternatively, in some applications, it might bedesirable to express the instant DS protein in specific plant tissuesand/or cell types, or during developmental stages in which they wouldnormally not be encountered.

Overexpression of the instant DS may be accomplished by firstconstructing a chimeric gene in which the DS coding region is operablylinked to a promoter capable of directing expression of a gene in thedesired tissues at the desired stage of development. For reasons ofconvenience, the chimeric gene may comprise promoter sequences andtranslation leader sequences derived from the same genes. 3' Non-codingsequences encoding transcription termination signals must also beprovided. The instant chimeric genes may also comprise one or moreintrons in order to facilitate gene expression.

Plasmid vectors comprising the instant chimeric genes can then beconstructed. The choice of a plasmid vector is dependent upon the methodthat will be used to transform host plants. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al., EMBO J.4:2411-2418 (1985); De Almeida etal., Mol. Gen. Genetics 218:78-86(1989)), and thus that multiple events must be screened in order toobtain lines displaying the desired expression level and pattern. Suchscreening may be accomplished by Southern analysis of DNA, Northernanalysis of MRNA expression, Western analysis of protein expression, orphenotypic analysis.

For some applications it may be useful to direct the DS protein todifferent cellular compartments or to facilitate their secretion fromthe cell. It is thus envisioned that the chimeric gene described abovemay be further modified by the addition of appropriate intracellular orextracellular targeting sequence to their coding regions. These includechloroplast transit peptides (Keegstra et al., Cell 56:247-253 (1989),signal sequences that direct proteins to the endoplasmic reticulum(Chrispeels et al., Ann. Rev. Plant Phys. Plant Mol. 42:21-53 (1991),and nuclear localization signal (Raikhel et al., Plant Phys.100:1627-1632 (1992). While the references cited give examples of eachof these, the list is not exhaustive and more targeting signals ofutility may be discovered in the future. It has been shown, for example,that the mature spinach RS is localized in chloroplasts. It has beenfurther demonstrated that antibodies directed against the purifiedrecombinant protein specifically interact with a polypeptide of theexpected size when spinach chloroplast extracts are subjected toSDS-PAGE and Western analysis.

It may also be desirable to reduce or eliminate expression of the DSgene in plants for some applications. In order to accomplish this,chimeric gene designed for antisense or co-suppression of DS can beconstructed by linking the genes or gene fragments encoding parts ofthese enzymes to plant promoter sequences. Thus, a chimeric genedesigned to express antisense RNA for all or part of DS can beconstructed by linking the DS gene or gene fragments in reverseorientation to plant promoter sequences. The co-suppression or antisensechimeric gene constructs could then be introduced into plants via wellknown transformation protocols to reduce or eliminate the endogenousexpression of DS gene products.

The DS protein produced in heterologous host cells, particularly in thecells of microbial hosts, can be used to prepare antibodies to theenzymes by methods well known to those skilled in the art. Theantibodies would be useful for detecting the instant DS protein in situin cells or in vitro in cell extracts. Preferred heterologous host cellsfor production of the instant DS protein are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct chimeric genes for production of the instant DS. Thesechimeric genes could then be introduced into appropriate microorganismsvia transformation to provide high level expression of the instant DSprotein.

Microbial host cells suitable for the expression of the instant DSenzymes include any cell capable of expression of the chimeric genesencoding these enzymes. Such cells will include both bacteria and fungiincluding, for example, the yeasts (e.g., Aspergillus, Saccharomyces,Pichia, Candida, and Hansenula), members of the genus Bacillus as wellas the enteric bacteria (e.g., Escherichia, Salmonella, and Shigella).Methods for the transformation of such hosts and the expression offoreign proteins are well known in the art and examples of suitableprotocols may be found in Manual of Methods for General Bacteriology(Gerhardt et al., eds., American Society for Microbiology, Washington,DC. (1994) or in Brock, T. D., Biotechnology: A Textbook of IndustrialMicrobiology, Second Edition, Sinauer Associates, Inc., Sunderland,Mass. (1989)).

Vectors or cassettes useful for the transformation of suitable microbialhost cells are well known in the art. Typically the vector or cassettecontains sequences directing transcription and translation of therelevant gene, a selectable marker, and sequences allowing autonomousreplication or chromosomal integration. Suitable vectors comprise aregion 5' of the gene which harbors transcriptional initiation controlsand a region 3' of the DNA fragment which controls transcriptionaltermination. It is most preferred when both control regions are derivedfrom genes homologous to the transformed host cell although it is to beunderstood that such control regions need not be derived from the genesnative to the specific species chosen as a production host.

Initiation control regions or promoters, which are useful to driveexpression of the gene encoding the DS enzyme in the desired host cellare numerous and familiar to those skilled in the art. Virtually anypromoter capable of driving these genes is suitable for the presentinvention including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1,PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful forexpression in Saccharomyces); AOX1 (useful for expression in Pichia);and lac, trp, lP_(L), lP_(R), T7, tac, and trc (useful for expression inEscherichia coli). Termination control regions may also be derived fromvarious genes native to the preferred hosts. Optionally, a terminationsite may be unnecessary, however, it is most preferred if included.

The instant DS protein can also be used as a tool to facilitate thedesign and/or identification of specific chemical agents that mightprove useful as fungicides, herbicides, or antibiotics. This could beachieved either through the rational design and synthesis of potentenzyme inhibitors that result from structural and/or mechanisticinformation that is derived from the purified instant fungal protein, orthrough random in vitro screening of chemical libraries. The DS proteincatalyzes an indispensable step in the synthesis of riboflavin in plantsand most microorganisms, and is required for the production of FAD andFMN, essential prosthetic groups for a number of important redoxenzymes. Consequently, it is anticipated that significant in vivoinhibition of any DS protein will severely cripple cellular metabolismand will likely result in the death of any organism that requires theendogenous production of riboflavin as its only source of the vitamin.

All or a portion of the nucleic acid fragments of the instant inventionmay also be used as probes for genetically and physically mapping thegenes that they are a part of, and as markers for traits linked toexpression of the instant DS. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes.

For example, the instant nucleic acid fragments may be used asrestriction fragment length polymorphism (RFLP) markers. Southern blots(Maniatis) of restriction-digested plant genomic DNA may be probed withthe nucleic acid fragments of the instant invention. The resultingbanding patterns may then be subjected to genetic analyses usingcomputer programs such as MapMaker (Lander et at., Genomics 1:174-181(1987)) in order to construct a genetic map. In addition, the nucleicacid fragments of the instant invention may be used to probe Southernblots containing restriction endonuclease-treated genomic DNAs of a setof individuals representing parent and progeny of a defined geneticcross. Segregation of the DNA polymorphisms is noted and used tocalculate the position of the instant nucleic acid sequence in thegenetic map previously obtained using this population (Botstein et al.,Am. J. Hum. Genet. 32:314-331 (1980)).

The production and use of plant gene-derived probes for use in geneticmapping is described by Bernatzky and Tanksley (Plant Mol. Biol.Reporter 4:37-41 (1986)). Numerous publications describe genetic mappingof specific cDNA clones using the methodology outlined above orvariations thereof. For example, F2 intercross populations, backcrosspopulations, randomly mated populations, near isogenic lines, and othersets of individuals may be used for mapping. Such methodologies are wellknown to those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al., Nonmammalian Genomic Analysis: APractical Guide, pp. 319-346, Academic Press (1996), and referencescited therein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequence may be used in direct fluorescence in situhybridization (FISH) mapping. Although current methods of FISH mappingfavor use of large clones (several to several hundred kb), improvementsin sensitivity may allow performance of FISH mapping using shorterprobes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification, polymorphismof PCR-amplified fragments (CAPS), allele-specific ligation, nucleotideextension reactions, Radiation Hybrid Mapping and Happy Mapping. Forthese methods, the sequence of a nucleic acid fragment is used to designand produce primer pairs for use in the amplification reaction or inprimer extension reactions. The design of such primers is well known tothose skilled in the art. In methods employing PCR-based geneticmapping, it may be necessary to identify DNA sequence differencesbetween the parents of the mapping cross in the region corresponding tothe instant nucleic acid sequences. This, however, is generally notnecessary for mapping methods. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes.

EXAMPLES

The present invention is farther defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usage and conditions.

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by Sambrook, J., Fritsch, E. F.and Maniatis, T. Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, 1989; and by T. J. Silhavy,M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, ColdSpring Harbor Laboratory Cold Press Spring Harbor, N.Y. (1984) and byAusubel, F. M. et al., Current Protocols in Molecular Biology, publishedby Greene Publishing Assoc. and Wiley-Interscience (1987).

Manipulations of genetic sequences were accomplished using the suite ofprograms available from the Genetics Computer Group Inc. (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wis.).

The meaning of abbreviations is as follows: "sec" means second(s), "min"means minute(s), "h" means hour(s), "d" means day(s), "EL" meansmicroliter, "mL" means milliliters, "L" means liters, "mM" meansmillimolar, "M" means molar, "mmol" means millimole(s).

Example 1 PCR-Cloning of Escherichia coli DS

Gene specific PCR primers were used to amplify the Escherichia coli DSgene from genomic DNA, while adding unique restriction sites to itsflanking regions for subsequent ligation into high copy number plasmids.The primers used for this purpose were based on the published DNAsequences of the Escherichia coli DS gene (GenBank accession numberX66720) and consisted of the following nucleotides:

Primer 1--(SEQ ID NO:3):

5'-ACT CAT TTA cca tgg CTC AGA CGC TAC TTT CCT C-3'

Primer 2--(SEQ ID NO:4):

5'-ATC TTA CTg tcg acT TCA GCT GGC TTT ACG CTC-3'

The underlined bases hybridize to the target gene, while lower caseletters indicate the restriction sites (NcoI or SalI) that were added tothe ends of the PCR primers.

Ampliflication of the DS gene was achieved using Primers 1 and 2, andgenomic DNA from Escherichia coli strain W3110 (Campbell et al., Proc.Natl. Acad. Sci. 75:2276-2284 (1978)). Primer 1 hybridizes at the startof the gene and introduces a NcoI site at the protein's initiationcodon, while Primer 2 hydridizes at the opposite end and provides a SalIsite just past the termination codon. The 100-μL PCR reactions contained˜100 ng of genomic DNA and both primers at a final concentration of 0.5μM. The other reaction components were provided by the GeneAmp PCRReagent Kit (Perkin Elmer), according to the manufacturer's protocol.Amplification was carried out in a DNA Thermocycler 480 (Perkin Elmer)for 28 cycles, each comprising 1 min at 94° C., 2 min at 53° C., and 2min at 72° C. Following the last cycle, there was a 7-min extensionperiod at 72° C. The PCR product was cut with NcoI and SalI, and ligatedinto similarly digested pGEM-5Zf (+) (Promega, Madison, Wis.). Thelatter was chosen as a suitable cloning vector since it lacks a NotIcleavage site after double-digestion with NcoI and SalI (see below). Theligation reaction mixture was used to transform Escherichia coli DH5αcompetant cells (GibcoBRL), and transformants were selected on LB mediasupplemented with 100 μg/mL ampicillin.

Plasmids harboring the cloned Escherichia coli DS gene were identifiedby restriction digestion analysis. Plasmid DNA was isolated from anumber of ampicillin-resistant colonies using the Wizard DNAPurification System (Promega, Madison, Wis.) and subjected to cleavagewith NcoI and SalI. The samples were analyzed by agarose gelelectrophoresis, and a representative plasmid for the gene, yielding aninsert of the correct size, was sequenced completely to verify theabsence of PCR errors. Apart from those nucleotides at the 5' and 3'ends that were intentionally altered for cloning purposes, the amplifiedEscherichia coli DS gene sequence was identical to that reported in theliterature.

Example 2 Insertional Inactivation of the Escherichia coli DS Gene

In order to create bacterial auxotrophs lacking the ability tosynthesize riboflavin, the cloned Escherichia coli DS gene was renderednonfunctional through insertional inactivation. Briefly, a unique NotIsite was introduced in the middle of the coding region of the targetgenes, and a DNA fragment that confers kanamycin resistance was ligatedinto the engineered site. The latter was provided by the commerciallyavailable Kan^(r) GenBlock cartridge (Pharmacia), that was modifiedthrough PCR to add NotI cleavage sites at both of its ends. Thismodification was accomplished using Primers 3 and 4 in a standard PCRreaction; the underlined portions hybridize to the Kan^(r) GenBlock, andlower case letters indicate the NotI cleavage sites.

Primer 3--(SEQ ID NO:5):

5'-AAC TAG ATC Agc ggc cgc AGC CAC GTT GTG TCT CAA A-3'

Primer 4--(SEQ ID NO:6):

5'-GAC AAA CAT Agc ggc cgc TGA GGT CTG CCT CGT GAA-3'

Following amplification, the modified Kanr GenBlock was cleaved withNotI, and the resulting fragment was purified by agarose gelelectrophoresis.

PCR primers were also used to introduce a unique NotI cleavage site inthe middle of the Escherichia coli DS gene. This was accomplishedthrough an application of the "inverse PCR" technique that is fullydescribed by Ochman, et al. in PCR Protocols: A Guide to Methods andApplications, (Innis et al., eds.) pp. 219-227, Academic Press, SanDiego, Calif., (1990). The targets for inverse PCR are usuallydouble-stranded circular DNA molecules. However, in contrast to otherPCR applications, the two primers are oriented away from each other suchthat their 3' ends are extended in opposite directions around the entirecircular template. If the primers are designed to hybridize immediatelyadjacent to each other, a linear DNA fragment is produced that includesthe entire vector sequence and has as its starting and stopping pointsthe original primer binding sites. The net result is analogous tolinearizing a circular plasmid at a specified location. By attachingappropriate nucleotide sequences to the non-hybridizing 5' ends of bothPCR primers, it is therefore possible to introduce a unique restrictionsite at any desired location within a circular template.

Primers 5 and 6 (which hybridize to nt 968-987 and nt 940-957 of the DNAsequence in GenBank accession number X66720) were designed to introducea NotI cleavage site in the middle of the Escherichia coli DS gene; thenucleotides that hybridize to the target gene are underlined, and NotIcleavage sites are indicated in lower case letters.

Primer 5--(SEQ ID NO:7):

5'-AAC TAG ATC Agc ggc cgc TGA CCG TAT TAC GAC-3'

Primer 6--LS (SEQ ID NO:8):

5'-GAC AAA CAT Agc ggc cgc GTA GTC ACA CCT TCA GCT-3'

The circular template for inverse PCR was the pGEM-5Zf (+) constructcontaining the Escherichia coli DS gene. The 100-μL PCR reactionscontained 0.5 ng of plasmid DNA and Primer 5 and Primer 6, both at afinal concentration of 0.5 μM. Amplification was carried out in a DNAThermocycler 480 (Perkin Elmer) for 30 cycles, each comprising 50 sec at94° C., 1 min at 55° C., and 3 min at 72° C. The PCR product was cleavedwith NotI and the resulting fragment was purified by agarose gelelectrophoresis; the excised band was of the expected size. Next, thepurified fragment was recircularized with T4 DNA ligase (Novagen) toregenerate a functional plasmid, and an aliquot of the ligation reactionmixture was used to transform Escherichia coli DH5α competent cells(GibcoBRL). Growth was selected for on LB media containing ampicillin(100 μg/mL), and plasmid DNA was isolated from a number of transformantsfor restriction digestion analysis with NotI, SalI, and NcoI. Arepresentative plasmid yielding the correct cleavage pattern with theseenzymes was selected for further manipulation.

To insert the kanamycin resistance gene, the plasmid construct describedabove was cleaved with NotI and purified by agarose gel electrophoresis.The fragment was then incubated with a 4-fold molar excess of themodified Kan^(r) GenBlock cartridge, and subjected to a standardligation reaction in the presence of T4 DNA ligase (Novagen). An aliquotof the ligation reaction mixtures was used to transform Escherichia coliDH5α competant cells (GibcoBRL), and growth was selected for on LBplates containing kanamycin (30 μg/mL) and ampicillin (100 μg/mL).Plasmids harboring the disrupted Escherichia coli DS gene wereidentified by restriction digestion analysis. The plasmids were cleavedwith NcoI and SalI, and were then subjected to agarose gelelectrophoresis to check for the presence of the inserted kanamycinresistance gene. A representative plasmid, yielding fragments of thecorrect size, was selected for further manipulation. DNA sequenceanalysis of this plasmid confirmed that the kanamycin resistance genehad been inserted at the correct location in the target gene.

Example 3 Generation of Escherichia coli DS Auxotroph

The insertionally inactivated Escherichia coli DS was liberated from theplasmid construct described above using NcoI and SalI and purified byagarose gel electrophoresis. The fragment was then introduced intoEscherichia coli strain ATCC 47002 (fully described in Balbas et al.,Gene 136:211-213 (1993), and isogenic with JC7623 (described byBachmann, B., in Escherichia coli and Salmonella typhimurium: Cellularand Molecular Biology (Niedhardt et al., eds.) p. 2466, American Societyof Microbiology, Washington, D.C. (1987)) by electroporatation using aBTX Transfector 100 (Biotechnologies and Experimental Research Inc.)according to the manufacturer's protocol. The choice of this strain asthe initial recipient for gene replacement was based on its wellestablished hyper-rec phenotype and related ability to undergo highfrequency double-crossover homologous recombination (Wyman et al., Proc.Nat. Acad. Sci. USA 82:2880-2884 (1985); Balbas et al., Gene 136:211-213(1993); Balbas et al., Gene 172:65-69 (1996)). Thus, it was anticipatedthat the insertionally inactivated Escherichia coli DS gene wouldefficiently replace its functional chromosomal counterpart in ATCC 47002under kanamycin selection.

Following electroporation, the transformed cells were resuspended in 1.0mL of S.O.C. media (GibcoBRL) that was supplemented with riboflavin (400μg/mL), and incubated for 1 h at 37° C. Kanamycin resistance was thenselected for on LB plates at 37° C. that contained both riboflavin (400μg/mL) and kanamycin (30 μg/mL); colonies appeared 24-48 h later.Phenotypic detection of the correct chromosomal integration event wasaccomplished through replica-plating experiments. Riboflavin auxotrophsresulting from double-crossover homologous recombination of thedisrupted target gene would be expected to be resistant to kanamycin,sensitive to ampicillin, and to exhibit growth only in the presence ofadded riboflavin. A representative bacterial colony exhibiting thisphenotype was selected for further study.

While ATCC 47002 is an excellent strain for creating Escherichia coli"knockouts", its multiple mutations in the recBCD loci render itincapable of propagating ColE1-type plasmids (Balbas et al., Gene172:65-69 (1996)). Consequently, the riboflavin auxotroph describedabove is not suitable for screening plasmid cDNA libraries by functionalcomplementation. In order to achieve this goal it was thereforenecessary to move the insertionally inactivated DS gene from thechromosome of ATCC 47002 to a suitable wildtype background. Thismanipulation was accomplished through generalized phage transductionusing P1_(vir) and standard methodologies as fully described by Miller,J. H., in Experiments in Molecular Genetics, pp. 201-205, Cold SpringHarbor Laboratory, Cold Spring Harbor, New York, (1972). Escherichiacoli W3110 (Campbell et al., Proc. Nat.l Acad. Sci. 75:2276-2284 (1978))was selected as the recipient strain for the insertionally inactivatedDS gene. Following phage transduction, bacterial growth was selected foron LB media that was supplemented with kanamycin (35 μg/mL) andriboflavin (400 μg/mL). Stable transductants harboring the disruptedEscherichia coli DS gene were then identified through replica-platingexperiments analogous to those described above for ATCC 47002. Thus,individual colonies were patched onto plates containing LB media, sodiumcitrate (7.5 mM), magnesium sulfate (1.5 mM), and kanamyacin (35 μg/mL),with or without riboflavin (400 μg/mL). The DS riboflavin auxotroph thatwas selected for further study and subsequent complementation cloning(see below) was only able to grow in the presence of added riboflavin,and was not resistant to ampicillin (100 μg/mL) or streptomycin (25μg/mL); sensitivity to streptomycin is characteristic of W3110, but notof ATCC 47002.

Example 4 Cloning of the Magnaporthe grisea DS Gene Through FunctionalComplementation

A Magnaporthe grisea cDNA expression library, in the NotI and SalIcleavage sites of the Lambda ZipLox vector (GibcoBRL), was prepared fromisolated MRNA using conventional methodologies (Maniatis supra), andsubjected to mass excision using the manufacturer's protocol. Uponexcision, the liberated cDNA inserts are contained in the plasmid vectorpZL1 which confers resistance to amipicillin, and allows theirexpression in Escherichia coli upon induction withisopropyl-1-thio-β-D-galactopyranoside (IPTG). The resulting mixture ofexcised plasmids was then electroporated into the Escherichia coli DSauxotroph (the W3110-derivative) using a BTX Transfector 100(Biotechnologies and Experimental Research Inc.) and the manufacturer'sprotocol. The transformed cells were selected for growth in the absenceof added riboflavin, on plates that contained B agar (LB mediacontaining sodium citrate (7.5 mM), magnesium sulfate (1.5 mM),kanamyacin (35 μg/mL), ampicillin (100 μg/mL), and IPTG (0.6 mM)).Following a 48-hr incubation period at 37° C., bacterial growth wasobserved and riboflavin-independant colonies were recovered atfrequencies of about 2.1×10⁻⁵. Plasmid DNA was isolated from arepresentative colony and subjected to further analysis; the selectedplasmid was able to transform the Escherichia coli DS auxotroph toriboflavin prototrophy at high frequency. The cDNA insert contained inthis plasmid was then sequenced completely on an ABI 377 automatedsequencer (Applied Biosystems), using fluorescent dideoxy terminatorsand custom-designed primers.

The approximately 1.3 kbp Magnaporthe grisea cDNA insert that rescuedthe Escherichia coli DS auxotroph clearly encodes a3,4-dihydroxy-2-butanone 4-phosphate synthase. The nucleotide sequenceof the open reading frame (ORF) for this protein and its predictedprimary amino acid sequence are set forth in SEQ ID NO:1 and SEQ IDNO:2, respectively.

Of the various microbial DS homologs that are shown in FIG. 1, theMangaporthe grisea protein (SEQ ID NO:2) show the greatest similarity tothe Vibrio harveyi and Saccharomyces cerevisiae proteins at the primaryamino acid sequence level (e.g., approximately 53% identity). Presently,the only known isolated fungal DS genes are that of Ashbya gossypii andSaccharomyces cerevisiae. Pairwise comparisons of the three fungal DSproteins affords the following percent identities:

52.5%--Mangaporthe grisea versus Saccharomyces cerevisiae.

48.1%--Mangaporthe grisea versus Ashbya gossypii.

59.9%--Saccharomyces cerevisiae versus Ashbya gossypii.

From the foregoing numbers, it is apparent the known fungal DS proteinshave a rather poor overall homology at their primary amino acid level.In addition, the Magnaporthe grisea DS is approximately only 44%identical to the corresponding protein of Eschericihia coli.

Example 5 Expression of Magnaporthe grisea DS in Escherichia coli

The cloned Magnaporthe grisea DS, identified in Example 4, was modifidedfor insertion into the T7 promoter-based Escherichia coli expressionvector, pET-24a (+) (Novagen), using primers 7 (SEQ ID NO:9) and 8 (SEQID NO:10). Primer 7 (5'-CTA CTC ATT TCA TAT GCC TTC CAC AGA CAG CAT-3')(SEQ ID NO:9) hydridizes to nt 1-20 of the Magnaporthe grisea DS (SEQ IDNO:1). It was designed to initiate protein synthesis in Escherichia coliat the normal starting point and incorporates for cloning purposes aunique NdeI site upstream from the initiator Met residue. Primer 8(5'-CAT CTT ACT AGA TCT TCA ACC CGA CCC ATT CGT C-3') (SEQ ID NO:10)hybridizes at the other end of the ORF to nt 684-702 and introduces aunique BglII site just past the protein's stop codon. The target for PCRamplification was the purified plasmid ontaining the cDNA insert for theMagnaporthe grisea DS. The predicted PCR product encodes the full-lengthMagnaporthe grisea DS with no modifications (SEQ ID NO:2).

Following amplification of the target gene, the PCR fragment was cleavedwith NdeI and BglII, and ligated into the NdeI and BamHI sites of thepolylinker region of pET-24a (+) (Novagen). The latter is a high-levelEscherichia coli expression vector that contains a T7 promoter and isselected for on kanamycin. An aliquot of the ligation reaction mixturewas then used to transform a plasmid-bearing derivative of Escherichiacoli BL21(DE3) using a BTX Transfector 100 (Biotechnologies andExperimental Research Inc.) according to the manufacturer's protocol.The plasmid contained in the BL21(DE3) cells that were used fortransformation was pGroESL (Goloubinoff et al., Nature 337:44-47(1985)), which confers resistance to chloramphenicol and constitutivelyoverexpresses the Escherichia coli GroEL and GroESproteins, twomolecular chaperones that are known from previous studies to assist inthe folding of certain other proteins. It was anticipated that thepresence of pGroESL in the bacterial host cells would improve theproduction of soluble Magnaporthe grisea DS which otherwise is foundexclusively in inclusion bodies. Following the transformation procedure,the cells were plated on LB media that contained both kanamyacin (50μg/ml) and chloramphenicol (60 μg/mL), and incubated at 37° C. to obtainsingle colonies. Clones harboring a Magnaporthe grisea DS insert inpET-24a (+) were identified through PCR reactions using individualresuspended colonies and primers 7 and 8. Following this procedure, arepresentative clone was selected for the production of recombinantprotein (see below) and its plasmid DNA was sequenced completely tocheck for PCR errors; none were found.

To express Magnaporthe grisea DS in Escherichia coli the straindescribed above was grown at 37° C. in LB media that contained kanamycin(50 μg/mL) and chloramphenicol (60 μg/mL). The cells were induced withIPTG (1 mM) at an A₆₀₀ nm of ˜1.0, and harvested 3 h later bycentrifugation. Magnaporthe grisea DS was well expressed in thebacterial host at levels exceeding 10% of the total protein. However,even with overexpression of GroEL and GroES, only about 25% of therecombinant protein was found to be soluble, and it was this materialthat was purifed as described below.

Example 6 Purification of Recombinant Magnaporthe grisea DS

Purification steps were performed at 04° C. Escherichia coli cellpellets containing (6.4 g wet wt.) Magnaporthe grisea DS were suspendedin 3 volumes of 50 mM Tris-HCl, 2 MM MgCl₂, pH 7.5 (Buffer P). Thesuspension was disrupted in a French pressure cell at 20,000 psi with 3passes though the cell with cooling on wet ice between each passage. Theresulting homogenate was centrifuged 20 min at 18,500 g and 5 mL of theresulting supernatant (53 mg/mL) was loaded onto a Pharmacia Q-SepharoseFast Flow column (1×5 cm) equilibrated with Buffer P. After washing thecolumn with 15 mL of Buffer P, the column was developed with a lineargradient (0-1M NaCl in Buffer P). DS activity eluted towards the end ofthe gradient. Active fractions were pooled (200 mg protein in 10 mL),concentrated to 5 mL and diafiltered 50× against 5 mM potassiumphosphate, 2.0 mM MgCl₂, pH 7.5 before loading onto a Calbiochemhydroxylapatite column (1×7 cm) equilibrated with 5 mM potassiumphosphate, 2.0 mM MgCl₂, pH 7.5. Active fractions eluted in the columnwash with equilibration buffer. They were concentrated to 2.5 mL (5.4mg/mL) and diafiltered 20× against 50 mM Tris-HCl, pH 7.5 before flashfreezing in liquid N₂ followed by storage at -80° C. until use. Thepurified DS was homogeneous as judged by SDS-PAGE and Coomassie bluestaining. Yield of DS was 13.5 mg based on an extinction coefficient at280 nm of 7270 M⁻¹ cm⁻¹ as calculated through the GCG Peptidesortprogram (Genetics Computer Group, Madison, Wis.). Yield of DS activity(1.5 μmole/min) was 35% of that in the crude homogenate. Specificactivity was 0.11 μmole/min/mg protein representing a 10-foldpurification over the crude homogenate.

Edman degradation of purified recombinant Magnaporthe grisea DS revealedthat its first 17 amino acids are identical to those of the proteinshown in SEQ ID NO:2. However, like a number of other proteins that areoverexpressed in Escherichia coli, its initiator Met residue is removedby the bacterial host.

Taking this into account, its predicted molecular mass is 24871.88daltons, which is in excellent agreement with the value that wasdetermined using electrospray ionization mass spectrometry (24870.7).More importantly, purified recombinant Magnaporthe grisea DS iscatalytically active. In the in vitro enzyme assay described below, itexhibited a turnover number of approximately 2.8 min⁻¹ (based onprotomer) at 25° C. By way of comparison, the reported turnover numberfor Escherichia coli DS at 37° C. is 0.66 min⁻¹ (Richter et al., J.Bacteriol. 174:4050-4056 (1992)) and Candida guilliermondii DS at 37° C.is 5.6 min⁻¹ (Volk et al., J. Biol. Chem. 265:19479-19485 (1990)).Assuming that the enzyme reaction is characterized by a Q10 (temperaturecoefficient) of 2, these observations suggest that the purifiedrecombinant Magnaporthe grisea DS is probably fully active.

Example 7 Assay for Measuring DS Enzyme Activity

Assays (0.125 mL) for DS activity were conducted in 96 well plates at25° C. Reactions (30 min) included 50 mM Tris-HCl, 10 mM MgCl₂, 5 mMriblse 5-phosphate, 0.25 unit of pentose phosphate isomerase and DS.Ribose 5-phosphate and pentose phosphate isomerase were used to generateDS substrate ribulose 5-phosphate in equilibrium with ribose 5-phosphatefor economy and purity reasons. In practice, ribose 5-phosphate andpentose phosphate isomerase may be replaced with the genuine DSsubstrate, ribulose 5-phosphate, with very similar results. Theimplication is that ribose 5-phosphate does not interfere with thecatalystic properties of DS. After 30 min, product3,4-dihydroxy-2-butanone 4-phosphate was determined by a modification ofa method which was developed for the determination of diacetyl(Mattessich et al. Anal. Biochem. 180:349-350 (1989)). To each reactionwas added 0.1 mL of a saturated creatine solution followed by 0.05 mL ofan a-naphthol solution (35 mg/mL in 1.0 N NaOH). Standard curves wereestablished with 0-36 nmol/0.125 mL of diacetyl or3,4-dihydroxy-2-butanone 4-phosphate and treating them as above for thereaction samples. Color was allowed to develop 30 min before readingabsorbances at 525 nm by using a Spectra Max Plus plate reader(Molecular Devices). For inhibition assays, ribose 5-phosphateconcentrations were changed to 0.5 mM and inhibitors were added to thereactions in 2 μL of DMSO.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES:  10                                         - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  702 bas - #e pairs                                               (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  DNA (genomic)                                    - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # Magnaporthe grisea DS                    - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #1:                          - - ATGCCTTCCA CAGACAGCAT ACCAAAGTCA AATTTTGACG CGATCCCAGA TG -             #TGATCCAA     60                                                                 - - GCATTCAAAA ACGGCGAGTT CGTAGTCGTG CTAGACGACC CCTCGCGCGA GA -            #ACGAAGCC    120                                                                 - - GACCTGATCA TCGCCGCCGA GTCGGTAACG ACGGAGCAGA TGGCCTTCAT GG -            #TGCGGCAC    180                                                                 - - TCGTCGGGGC TGATCTGCGC CCCGCTGACG CCGGAGCGCA CCACCGCCCT CG -            #ACCTGCCG    240                                                                 - - CAGATGGTGA CGCACAACGC CGACCCGCGC GGCACCGCCT ACACCGTCTC GG -            #TCGACGCC    300                                                                 - - GAGCACCCCT CCACCACCAC CGGCATAAGC GCGCACGACC GCGCCCTCGC CT -            #GCCGCATG    360                                                                 - - CTCGCCGCTC CCGACGCCCA GCCCTCGCAC TTTCGCCGCC CGGGCCACGT CT -            #TCCCCCTC    420                                                                 - - CGCGCCGTCG CGGGCGGCGT CAGGGCCCGC AGGGGCCATA CCGAGGCCGG TG -            #TAGAGCTG    480                                                                 - - TGCAGGCTGG CGGGCAAGAG GCCCGTCGCT GTCATCAGCG AGATTGTCGA CG -            #ATGGGCAA    540                                                                 - - GAGGTCGAGG GCCGGGCCGT GCGTGCCGCG CCGGGCATGT TGAGGGGTGA TG -            #AGTGCGTG    600                                                                 - - GCGTTTGCGC GGAGGTGGGG CCTCAAGGTC TGCACTATCG AGGATATGAT TG -            #CCCATGTG    660                                                                 - - GAAAAGACAG AGGGGAAGCT CGAGACGAAT GGGTCGGGTT GA    - #                      - # 702                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  233 ami - #no acids                                              (B) TYPE:  amino aci - #d                                                     (C) STRANDEDNESS: Not R - #elevant                                            (D) TOPOLOGY: Not Relev - #ant                                       - -     (ii) MOLECULE TYPE:  protein                                          - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # Magnaporthe grisea DS                    - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #2:                          - - Met Pro Ser Thr Asp Ser Ile Pro Lys Ser As - #n Phe Asp Ala Ile Pro      1               5   - #                10  - #                15               - - Asp Val Ile Gln Ala Phe Lys Asn Gly Glu Ph - #e Val Val Val Leu Asp                  20      - #            25      - #            30                   - - Asp Pro Ser Arg Glu Asn Glu Ala Asp Leu Il - #e Ile Ala Ala Glu Ser              35          - #        40          - #        45                       - - Val Thr Thr Glu Gln Met Ala Phe Met Val Ar - #g His Ser Ser Gly Leu          50              - #    55              - #    60                           - - Ile Cys Ala Pro Leu Thr Pro Glu Arg Thr Th - #r Ala Leu Asp Leu Pro      65                  - #70                  - #75                  - #80        - - Gln Met Val Thr His Asn Ala Asp Pro Arg Gl - #y Thr Ala Tyr Thr Val                      85  - #                90  - #                95               - - Ser Val Asp Ala Glu His Pro Ser Thr Thr Th - #r Gly Ile Ser Ala His                  100      - #           105      - #           110                  - - Asp Arg Ala Leu Ala Cys Arg Met Leu Ala Al - #a Pro Asp Ala Gln Pro              115          - #       120          - #       125                      - - Ser His Phe Arg Arg Pro Gly His Val Phe Pr - #o Leu Arg Ala Val Ala          130              - #   135              - #   140                          - - Gly Gly Val Arg Ala Arg Arg Gly His Thr Gl - #u Ala Gly Val Glu Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Cys Arg Leu Ala Gly Lys Arg Pro Val Ala Va - #l Ile Ser Glu Ile        Val                                                                                             165  - #               170  - #               175             - - Asp Asp Gly Gln Glu Val Glu Gly Arg Ala Va - #l Arg Ala Ala Pro Gly                  180      - #           185      - #           190                  - - Met Leu Arg Gly Asp Glu Cys Val Ala Phe Al - #a Arg Arg Trp Gly Leu              195          - #       200          - #       205                      - - Lys Val Cys Thr Ile Glu Asp Met Ile Ala Hi - #s Val Glu Lys Thr Glu          210              - #   215              - #   220                          - - Gly Lys Leu Glu Thr Asn Gly Ser Gly                                      225                 2 - #30                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  34 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION:  /desc - # = "primer"                               - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # 5' E. coli DS primer                     - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #3:                          - - ACTCATTTAC CATGGCTCAG ACGCTACTTT CCTC       - #                  -      #        34                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  33 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION:  /desc - # = "primer"                               - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # 3' E. coli DS primer                     - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #4:                          - - ATCTTACTGT CGACTTCAGC TGGCTTTACG CTC       - #                  -      #         33                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  37 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  other nucleic aci - #d                                    (A) DESCRIPTION:  /desc - # = "primer"                               - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # 5' E. coli DS primer                     - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #5:                          - - AACTAGATCA GCGGCCGCAG CCACGTTGTG TCTCAAA      - #                      - #      37                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  36 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  other nucleic aci - #d                                    (A) DESCRIPTION:  /desc - # = "primer"                               - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # E. coli DS primer                        - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #6:                          - - GACAAACATA GCGGCCGCTG AGGTCTGCCT CGTGAA      - #                  -     #       36                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  33 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  other nucleic aci - #d                                    (A) DESCRIPTION:  /desc - # = "primer"                               - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # E. coli DS primer                        - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #7:                          - - AACTAGATCA GCGGCCGCTG ACCGTATTAC GAC       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  36 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  other nucleic aci - #d                                    (A) DESCRIPTION:  /desc - # = "primer"                               - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # E. coli DS primer                        - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #8:                          - - GACAAACATA GCGGCCGCGT AGTCACACCT TCAGCT      - #                  -     #       36                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  33 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  other nucleic aci - #d                                    (A) DESCRIPTION:  /desc - # = "primer"                               - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # 5' E. coli DS primer                     - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #9:                          - - CTACTCATTT CATATGCCTT CCACAGACAG CAT       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  34 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  other nucleic aci - #d                                    (A) DESCRIPTION:  /desc - # = "primer"                               - -    (iii) HYPOTHETICAL:  NO                                                - -     (iv) ANTI-SENSE:  NO                                                  - -     (vi) ORIGINAL SOURCE:                                                          (C) INDIVIDUAL ISOLATE: - # 3' E. coli DS primer                     - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #10:                         - - CATCTTACTA GATCTTCAAC CCGACCCATT CGTC       - #                  -      #        34                                                                   __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid fragment encoding a DSenzyme selected from the group consisting of:(a) an isolated nucleicacid fragment encoding the amino acid sequence set forth in SEQ ID NO:2,or an enzymatically active fragment thereof; (b) an isolated nucleicacid fragment that hybridizes to the nucleic acid fragment of (a) underthe conditions of 0.1× SSC, 0.1% SDS at 65° C.; (c) an isolated nucleicacid fragment encoding a polypeptide having at least 60% identity withthe amino acid sequence set forth in SEQ ID NO:2; and (d) an isolatednucleic acid fragment that is complementary to (a), (b), or (c).
 2. Theisolated nucleic acid fragment of claim 1 as set forth in SEQ ID NO:1.3. The isolated nucleic acid fragment of claim 1 encoding a fungal DSenzyme obtained from Magnaporthe grisea.
 4. A chimeric gene comprisingthe isolated nucleic acid fragment of claim 1 operably linked tosuitable regulatory sequences.
 5. A transformed host cell comprising ahost cell and the chimeric gene of claim
 4. 6. The transformed host cellof claim 5 wherein the host cell is a plant cell.
 7. The transformedhost cell of claim 5 wherein the host cell is Escherichia coli.
 8. Amethod of altering the level of expression of a DS enzyme in a host cellcomprising:(a) transforming a host cell with the chimeric gene of claim4; and (b) growing the transformed host cell of step (a) underconditions that are suitable for expression of the chimericgene,resulting in production of altered levels of a DS enzyme relativeto expression levels of an untransformed host cell.
 9. A method ofobtaining a nucleic acid fragment encoding a fungal DS enzymecomprising:(a) probing a cDNA or genomic library with 20 or morecontigious nucleotides isolated nucleic acid fragment of claim 1; (b)identifying a DNA clone that hybridizes with the isolated nucleic acidfragment of claim 1; and (c) sequencing the cDNA or genomic fragmentthat comprises the clone identified in step (b),wherein the sequencedcDNA or genomic fragment encodes a fungal DS enzyme.
 10. A method ofobtaining a nucleic acid fragment encoding a fungal DS comprising:(a)synthesizing at least one oligonucleotide primer corresponding to 20 ormore contigious nucleotides or the isolated nucleic acid fragment ofclaim 1; and (b) amplifying a cDNA insert present in a cloning vectorusing the oligonucleotide primer of step (a),wherein the amplified cDNAinsert encodes a fungal DS enzyme.
 11. A nucleic acid fragment preparedby the method of claim 9 or 10.